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PHA111 > DNA replication > Flashcards

DNA replication Flashcards

1
Q

outline briefly gene expression?

A
  1. transcription - nucleotide sequence of gene copied into RNA
  2. translation - nucleotide sequence of RNA directs order —> AA —> protein
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2
Q

describe the Hersey Chase (1952) experiment?

A

tried to prove dna was genetic material

  • viruses grown in one of two isotopic medium to radioactively label specific viral component
  • virus gown in radioactive sulphur (35S) had radio labelled proteins (sulphur present in proteins NOT dna)
  • virus grown in radioactive phosphorous (32P) had radio labelled dna (phosphorous present in dna not proteins)
  • virus then infected bacterium (e.coli)
  • virus + bacteria separated by centrifugation
  • larger bacteria = solid pellet
  • smaller viruses left in supernatant
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3
Q

what did the Hersey Chase experiment prove?

A
  • bacterial pellet found = radioactive when infected by 32P-viruses (DNA) + NOT 35S virus (protein)
  • DNA NOT PROTEIN = genetic material
  • DNA transferred to bacteria
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4
Q

what does semi-conservative dna replication give?

A

2 duplex DNA mol each of which contain one old + one new strand

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5
Q

what does conservative dna replication give?

A

2 duplexes

one has 2 old strands and second has 2 new strands

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6
Q

what does dispersive dna replication give?

A

2 daughter duplexes

both contain mixture of old and new strands

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7
Q

experiments by meselsohn + stahl (1958) proved which dna replication model?

A

semi - conservative

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8
Q

describe the meselsohn + stahl experiment?

A
  • grow bacteria in 15N (heavy) medium
  • transfer some bacteria to 14N (light) medium —> bacteria grows
  • take samples after 0, 20 and 40 mins
  • before bacteria reproduce for 1st time in light medium (0mins), all DNA (parental) = heavy
  • after 2 gen, half dna = intermediate and half = light only (no only heavy dna)
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9
Q

how long is one round + 2 rounds of replication?

A

one round = 20 mins

two rounds = 40 mins

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10
Q

what was the conclusion obtained from the meselsohn + stahl experiment?

A

pattern only observed if each dna molecule has template from parental dna —> dna replication = semi-conservative

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11
Q

replication is initiated at an ‘origin of replication’ that gives rise to two…

A

replication forks

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12
Q

what is a replication fork?

A

branch point in replication eye at which DNA synthesis occurs

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13
Q

what may a replication bubble contain?

A

1/2 replication forks (unidirectional/bidirectional replication)

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14
Q

what is dna replication almost always?

A

bidirectional

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15
Q

how many replication origins do prokaryotic and bacteriophages have?

A

1

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16
Q

what did reniji Okazaki elucidate?

A

semi-discontinuous model of dna replication

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17
Q

prokaryotic replication initiation - dnaA proteins?

A
  • 4 copies of 9-bp sequence that bind dnaA proteins
  • once all binding sites full ——> cooperatively recruit more dnaA proteins = dnaA barrel
  • helps open up local AT rich region of dna via TORSIONAL STRESS
  • pair of replication forks generated
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18
Q

prokaryotic replication initiation - dnaB proteins?

A
  • dnaB recruited to replication fork to initiate formation of pre-priming complex
  • dnaB = helicase enzyme: breaks HB
  • open dna strands covered with SSBs (single stranded binding proteins)
  • SSBs stop strands re-annealing + protect dna from attach by free radicals + nuclease enzymes
  • initiation complete
  • next phase = elongation
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19
Q

describe the polymerase reaction?

A

synthesis of new strand carried out by dan-dependent DNA polymerase enzymes

  • dna generated in 5’ to 3’ direction
  • polymerase moves along template strand in 3’ to 5’ direction
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20
Q

describe prokaryotic elongation?

A
  • primase enzyme (DnaG) binds near to heliase
  • starts to synthesise RNA primer on primosome
  • single stand binding protein stabilise lagging strand
  • dna polymerase III holoenzyme clamps to leading strands + synthesises dna
21
Q

semi-discontinuous replication

A

carried out by dna polymerase in 5’ to 3’ direction

  • polymerase enzyme inserts 5’ nucleotide first + extends towards 3’ end

template dna mol always used in 3’ to 5’ direction

22
Q

what is the lagging strand generated via?

A

synthesis of multiple Okazaki fragments

23
Q

the lagging strand is generated in ….. direction to movement of replication fork

A

opposite

24
Q

all dna replication is started with a short rna primer.

what does this allow?

A

proofreading of newly synthesised strand

25
Q

how are primers removed?

A

by the exonuclease activity of the polymerase complex

26
Q

what allows both strands of dna to be replicated at the same time?

A

2 dna polymerase enzymes = tethered together - each replicating one strand

27
Q

what is a replisome?

A

leading strand continuously in 5’ to 3’ direction

lagging strand discontinuously in 5’ to 3’

28
Q

describe the nature of the lagging strand?

A

looped over top of replisome so both polymerases move in same direction

29
Q

which enzyme proceeds ahead of the replisome?

A

topoisomerase

30
Q

how are the Okazaki fragments joined?

A

by ligase enzymes (ligation)

  • dna polymerase III replaced with another polymerase (pol I)
  • removes rna primer on Okazaki fragment before ligation
31
Q

why are topoisomerases needed to alleviate the problems within dna elongation?

A
  • replication fork can only progress short distance before there = topological prob
  • double helix needs rotation as helix = opened to stop over-winding ahead of replication fork
32
Q

what are the 2 types of topoisomerases?

A

Type I - introduces real in 1 strand —> pass other strand through + then reseal break

Type II - breaks both strands —> passes double helix through gap —> reseals break

33
Q

how the breaks in dna attached and why?

A

covalently

so ends don’t loosen

34
Q

in e.coli where do the 2 replicons meet?

A

180 degrees away from the origin

35
Q

what is in place to make sure the replicon meed at a specific point?

A

regulatory mechanism

if 1 gets there first, it will wait for the 2nd to arrive before signalling completion of dna replication

36
Q

what do specific terminator sequences signal?

A

that replication is approaching stop sequence

37
Q

what happens if the replicon meets a transcription bubble (mRNA synthesis)?

A

will wait and not overtake

38
Q

describe eukaryotic replication forks?

A
  • slower than prokaryotic forks
  • move at around 50bp per second
  • multiple forks needed due to size (50,000-100,000) per mammalian cell
39
Q

in eukaroytes, clusters of how many replicons will initiate simultaneously at defined time in S-phase based upon mitogen availability?

A

20-50

40
Q

early S-phase clusters

A

euchromatin- transcriptionally active dna

41
Q

late S-phase clusters

A

heterochromatin-transcriptionally silent dna

42
Q

centromeric and telomeric dna

A

replicated last

43
Q

how is the sequence in yeast bound?

A
  • by origin replication complex (ORC)
  • activated by cyclin dependent kinases (CDKs)
  • facilitates/initiates opening of dna duplex
44
Q

how many times can eukaryotic dna replicons initiate?

A

once per cell

  • so dna = fully + controllably replicated before cell division
  • limits introduction of mutations
45
Q

what is needed for the initiation of dna replication?

A
  • protein licensing factor complex
  • inactivated after use
  • only able gain access to nucleus when nuclear envelope dissolves in mitosis
46
Q

basically the origin is..

A
  • identified (binding of ORC)
  • set up (binding of initiation factors)
  • checked
  • initiation
47
Q

why can the ends of chromosomes not be replicated by semi-discontinuous replication?

A

no dna to elongate once rna primer removed from 5’ -end of lagging strand

could lead to loss of genetic material

48
Q

how can u overcome this problem?

A

eukaryotic chromosomes have hundreds of copies of non-coding repeat sequence

3’ end hangs over 5’ end

telomerase associates with short rna mol that = partially complementary to this sequence

rna acts as template for addition of repeats to 3’ end overhangs

comp strand = synthesised by normal lagging strand synthesis —> leaves 3’ overhang

49
Q

where is telomerase activity repressed and why?

A
  • in somatic cells

leads to gradual loss of dna + shortening of chromosomes

may cause problems in ageing process

PHA111 (30 decks)

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